During the current reporting period the major focus of this project has been the discovery and characterization of a new recessively inherited autoinflammatory disease. We also published a paper on the use of the long-acting interleukin-1 blocker, rilonacept, in FMF, and we completed a collaboration with Dr. Raphaela Goldbach-Mansky of NIAMS to study the genetic basis of chronic atypical neutrophilic dermatitis with lipodystrophy and elevated temperature (CANDLE). Deficiency of ADA2, a New Autoinflammatory Disease Beginning about 10 years ago, we have seen a total of 5 patients at the NIH Clinical Center with intermittent fevers, recurrent lacunar strokes, elevated acute phase reactants, livedoid rash, hepatosplenomegaly, and hypogammaglobulinemia. In 4 patients skin biopsies of livedoid lesions showed an inflammatory infiltrate of neutrophils, macrophages, and T lymphocytes; in one patient necrotizing vasculitis was present in the deep dermis. Magnetic resonance imaging showed evidence for small vessel acute brain ischemia and old ischemic strokes in the deep nuclei of the brain. Several stroke events were hemorrhagic or underwent hemorrhagic transformation, although the interpretation is clouded by the concomitant use of anti-platelet agents and/or warfarin. Four patients had hepatosplenomegaly, and one had documented portal hypertension. IgM levels were consistently low in all 5. We performed whole-exome sequencing in the first 3 patients and their unaffected parents, filtering the data under both recessive and de novo dominant models of inheritance. A single common candidate gene was identified only under the recessive model. We found compound heterozygous predicted deleterious mutations in CECR1 (chromosome 22), encoding adenosine deaminase type 2 (ADA2). Two of the patients shared the p.Tyr453Cys mutation; one patient had a 28 kb genomic deletion that included the 5 UTR and exon 1 of CECR1. Among the 3 patients we identified a total of 5 CECR1 mutations. We then sequenced CECR1 in the 2 remaining NIH patients and a patient from the UK with a similar phenotype. All 3 were compound heterozygous for CECR1 mutations, including 3 novel mutations. We subsequently performed CECR1 candidate gene sequencing on 3 Turkish patients, 2 brothers who carried the diagnosis of polyarteritis nodosum (PAN) and a third patient with necrotizing small vessel vasculitis. All 3 were homozygous for the p.Gly47Arg mutation. Among these 9 patients we identified 9 predicted deleterious ADA2 mutations, including 2 null mutations. Mutations in the related protein ADA1 cause severe combined immunodeficiency disease (SCID) because of the failure to catalyze the conversion of adenosine (and deoxyadenosine) to inosine (and deoxyinosine), and the consequent accumulation of toxic metabolites. ADA2 also catalyzes this reaction, but with a lower affinity for adenosine. Using enzyme assays that distinguish ADA1 from ADA2 activity, we found that patients had nearly absent ADA2 activity, but normal ADA1 activity, in the blood. Direct assays of adenosine/deoxyadenosine metabolites were negative. Assays of serum cytokines, peripheral T cell function, and peripheral B cells demonstrated relatively modest defects in the B cell compartment. Although there is no clear CECR1 ortholog in the mouse, there are two paralogs in the zebrafish. Morpholino knockdown of one of the zebrafish paralogs (cecr1b) caused intracranial hemorrhages and neutropenia, phenotypes that were rescued by wild type but not mutant human CECR1. These data, in conjunction with data from the literature indicating that ADA2 may be the prototype for a family of adenosine deaminase-derived growth factors, prompted a closer examination of the role of ADA2 in endothelial and leukocyte development in patients. We found that ADA2 is not produced in human endothelial cells. From the literature it is known that ADA2 is expressed in myeloid cells. Immunohistochemistry of skin and brain biopsies from patients demonstrates both endothelial damage and activation; staining for IL-1beta, TNF-alpha, and iNOS indicates inflammation. Using shRNA to silence the expression of ADA2 in myeloid U937 cells, and studies of patient monocytes stimulated with defined growth factors, we found that ADA2 deficiency is associated with a skewing of monocytes to the more inflammatory M1 subpopulation. When shRNA treated U937 cells, or untreated patient monocytes, were cocultured with endothelial cell layers, they induced damage, relative to control U937 cells or healthy control monocytes. We conclude that loss-of-function mutations in CECR1 cause a spectrum of vascular and inflammatory phenotypes ranging from early-onset recurrent stroke to systemic vasculopathy and/or vasculitis. Taken together, our zebrafish and patient data suggest that ADA2 deficiency may diminish endothelial integrity while polarizing macrophage/monocyte subsets towards pro-inflammatory cells, thus establishing a vicious circle of vasculopathy and inflammation. Rilonacept in FMF Currently there is no proven alternative for FMF patients resistant to or intolerant of colchicine. Previous data have implicated interleukin-1 as a key proinflammatory cytokine in FMF. We assessed the efficacy and safety of rilonacept, an interleukin-1 decoy receptor, using a double-blind, single-subject alternating treatment design. Subjects over 4 years old with at least 1 monthly attack despite adequate doses of colchicine, or who were colchicine intolerant, were recruited at 6 U.S. sites. Subjects were randomized to 1 of 4 treatment sequences that included two 3-month courses of rilonacept 2.2 mg/kg (max 160 mg) by weekly SC injection, and two 3-month courses of placebo. Eight males and 6 females with a mean age of 24.4 years (SD, 11.8) were randomly assigned. Among 12 participants who completed 2 or more courses, the rilonacept-placebo attack risk ratio was 0.59 (SD, 0.12) (equal-tail 95% credible interval, 0.39 to 0.85). The median number of attacks per month was 0.77 (0.18 and 1.20 attacks in the first and third quartiles, respectively) with rilonacept versus 2.00 (0.90 and 2.40, respectively) with placebo (median difference, -1.74 95% CI, -3.4 to -0.1, P = 0.027). There were more treatment courses of rilonacept without attacks (29% vs. 0%; P = 0.004), and with a decrease in attacks of greater than 50% compared with the baseline rate during screening (75% vs. 35%, P = 0.006) than with placebo. However, the duration of attacks did not differ between placebo and rilonacept (median difference, 1.2 days (-0.5 and 2.4 days in the first and third quartiles, respectively); P = 0.32). Injection site reactions were more frequent with rilonacept (median difference, 0 events per patient treatment month (medians of -4 and 0 in the first and third quartiles, respectively); P = 0.047), but no differences were seen in other adverse events. We conclude that rilonacept reduces the frequency of FMF attacks and is a treatment option for colchicine-resistant or intolerant FMF. Mutational Analysis in CANDLE During a previous reporting period our laboratory participated in a study that identified mutations in a proteasome protein, PSMB8, in patients with CANDLE. Nevertheless, there remained patients with the CANDLE phenotype with only a single demonstrable PSMB8 mutation, and other patients with no demonstrable PSMB8 mutations. By systematically performing Sanger sequencing on the genes encoding various proteasome components, in conjunction with whole-exome sequencing to exclude other possible candidates, we identified patients who are double heterozygous for a missense mutation in a gene encoding an inducible proteasome subunit (PSMB8, PSMB9) and a constitutive subunit (PSMA3, PSMB4), suggesting a digenic model of inheritance. A manuscript describing these findings is in preparation.